Taxol is a natural product first isolated from the Pacific Yew tree, Taxus brevifolia, by Wani et al. (1971, J. Am. Chem. Soc. 93: 2325). Among the antimitotic agents, taxol, which contains a diterpene carbon skeleton, exhibits a unique mode of action on microtubule proteins responsible for the formation of the mitotic spindle. In contrast with other antimitotic agents such as vinblastine or colchicine, which prevent the assembly of tubulin, taxol is the only plant product known to inhibit the depolymerization process of tubulin. This prevents the cell replication process and taxol has been shown to have significant antineoplastic and anticancer effects in drug-refractory ovarian cancer. Taxol has shown excellent antitumor activity in a wide variety of tumor models such as the B16 melanoma, L1210 leukemias, MX-1 mammary tumors, and CX-1 colon tumor xenografts. Several recent press releases have termed taxol as the new anticancer wonder-drug. The poor aqueous solubility of taxol has, however, remained a setback in human clinical trials, and currently used formulations require a cremaphore to solubilize the drug. The human clinical dose range is 200-500 mg and requires about one liter of fluid given intravenously using the cremaphore. In phase I clinical trials taxol itself did not show excessive toxic effects but severe allergic reactions were caused by the emulsifiers administered to solubilize the drug.
The general chemical structure of taxol is shown in FIG. 1 in which R.sub.1 =R.sub.2 =OH. Potential sites for modification of the drug are at the hydroxyls on the 1, 7, and 2' Carbon atoms. The 1-hydroxyl is sterically hindered and nonreactive, the 2'-hydroxyl is the most reactive, followed by the 7-hydroxyl which is also sterically hindered. Thus the modification of taxol to increase its water-solubility has revolved around the modification of the 2'- and the 7-hydroxyls. Studies have reported that the C-13 ester side chain and the 2'-hydroxyl group on the side chain are essential for biological activity. Mellado et al. (1984; Biochem. Biophys. Res. Commun. 124:329-336) have reported the synthesis of 2'-acetyl, 7-acetyl, and 2',7-diacetyl taxol. An acetyl at the 2'position resulted in a loss in ability to promote microtubule assembly. Taxol and 7-acetyl taxol were similar in their ability to alter cell proliferation and microtubule polymerization. These observations suggest that the 2'- and 7-positions are suitable for structural modifications, the 2'-position as a site for reversible derivatization (or formation of a prodrug) and the 7-position for analogue/prodrug modifications.
A number of chemically modifed taxols with enhanced water-solubilities have been developed. Among them are the sulfonated derivatives (Kingston et al., 1991; U.S. Pat. No. 5,059,699), and amino acid esters (Mathew et al., 1992; J. Med. Chem. 3B:145-151) which show significant biological activity. However, the delivery of taxol attached to a macromolecular or polymeric water-soluble carrier has not been considered. Nathan et al. (1990; Polymer Preprints 31: 213-214) have described a polyethylene glycol (PEG) chain-extended with amino acids such as lysine, to produce a polymer which has pendant carboxylic acid groups that may be used to attach biologically active molecules. However, no mention is made of the immobilization of taxol, or the attachment of a water-insoluble drug to such a carrier in order to deliver it in a soluble form.
In the present invention, to deliver taxol in a water-soluble form we have used a water-soluble polymer to which the drug is bound, the resultant polymer-drug conjugate being soluble. Water-soluble polymers such as PEG, have been investigated extensively in recent years for use as nontoxic, biocompatible, protein repulsive, noninflammatory, and nonimmunogenic modifiers for drugs, proteins, enzymes, and surfaces of implanted materials. These characteristics have been variously attributed to a combination of properties of these polymers, e.g., nonionic character, water solubility, backbone flexibility, and volume exclusion effect in solution or when immobilized at a surface. The solubility of PEG in water as well as a number of common organic solvents facilitates its modification by a variety of chemical reactions and makes it amenable for binding water-insoluble or poorly water-soluble molecules and rendering them water-soluble.
The preparation of a reversible PEG-taxol derivative at the 2'- and/or 7-position on taxol serves as useful aqueous-soluble prodrug. A nonreversible PEG derivative on the 7-position of taxol serves as a useful water-soluble drug analogue.
Advantages of delivering the drug attached to a water-soluble polymer as described in the present invention are many fold. The number of drug molecules per polymer molecule can be controlled; the circulation time of the drug can be varied by adjusting a number of variables including molecular weight of the polymeric carrier, the type of linkage between the drug and polymer, i.e., some linkages are hydrolyzed at much faster rates than others; large increases or decreases in blood levels of the drug may be avoided in favor of more gradual and sustained levels obtained, by continuous release of the drug from a polymeric carrier; and the hydrolysis of the drug-polymer conjugate results in the formation of the original biologically active drug and the innocuous water-soluble polymer that is excreted from the body.